It is known that proteins in vivo are cleared by various mechanisms, including degradation by serum proteases, elimination through the kidneys, or clearance by receptors. Thus, various attempts have been made to avoid such protein clearance mechanisms to increase the half-life of physiologically active proteins to thereby enhance their therapeutic efficacy. Particularly, studies have been conducted on protein conjugates that comprise a polyethylene glycol polymer (PEG), albumin, fatty acid or an antibody Fc fragment (constant region) linked to a protein in order to increase the half-life of the protein. Such studies aim to covalently link this material to a physiologically active protein in order to increase the serum half-life of the physiologically active protein and shorten the interval of drug administration to thereby increase the patient's convenience. Particularly, in order to stabilize proteins and inhibit their contact with proteases and their elimination through the kidneys, a method of chemically attaching highly soluble polymers such as PEG to the surface of the protein drugs is used. In the case of this method, it is known that a polymer binds non-specifically to a specific site or various sites of a target protein to increase the solubility of the protein, stabilize the protein and prevent the hydrolysis of the protein, and furthermore, causes no particular side effects (Sada et al., J. Fermentation Bioengineering 71: 137-139, 1991). However, this method has problems in that even though the PEG linked to the physiologically active protein can increase the stability of the protein, it significantly reduces the titer of the protein, and as the molecular weight of the PEG increases, its reactivity with the protein decreases, resulting in a decrease in the yield. In addition, when a specific amino acid residue of a protein is modified with fatty acids, the modified fatty acid binds reversibly to serum albumin to increase the serum half-life of the protein, but the half-life is about one day to one week, indicating that the increasing the half-life is not so significant. In addition, there is a disadvantage in that the physiologically active protein reversibly dissociated from albumin is easily eliminated through the kidneys.
For these reasons, efforts have been made to use immunoglobulin fragments to increase the half-life of physiologically active materials, including proteins. Particularly, studies have been actively conducted to increase the stability of therapeutic proteins by fusing the therapeutic proteins with such immunoglobulin Fc fragments.
It is known to express interferon (Korean Patent Laid-Open Publication No. 2003-9464), interleukin-4 receptor, interleukin-7 receptor or erythropoietin receptor (Korean Patent No. 249572) as a fusion with an immunoglobulin Fc fragment in a mammal by a genetic recombination method. Also, International Patent Publication No. WO 01/03737 discloses a fusion protein comprising a cytokine or growth factor linked to an immunoglobulin Fc fragment via an oligopeptide linker. Moreover, U.S. Pat. No. 5,116,964 discloses a fusion protein comprising an LHR (lymphocyte cell surface glycoprotein) or CD4 protein fused to the amino or carboxy terminal end of an immunoglobulin Fc fragment by a genetic recombination method, and U.S. Pat. No. 5,349,053 discloses a fusion protein of IL-2 with an immunoglobulin Fc fragment. In addition, examples of Fc fusion proteins prepared by genetic recombination methods include a fusion protein of interferon-beta or its derivative with an immunoglobulin Fc fragment (International Patent Publication No. WO 00/23472), a fusion protein of IL-5 receptor with an immunoglobulin Fc fragment (U.S. Pat. No. 5,712,121), a fusion protein of interferon-alpha with an immunoglobulin G4 Fc fragment (U.S. Pat. No. 5,723,125), and a fusion protein of CD4 protein with an immunoglobulin G2 Fc fragment (U.S. Pat. No. 6,451,313). Additionally, U.S. Pat. No. 5,605,690 relates to the modification of amino acid residues in an immunoglobulin Fc fragment and discloses a TNFR-IgG1 Fc fusion protein prepared by a genetic recombination method using an Fc fragment obtained by modifying the amino acid residues of a particular complement-binding site or a receptor-binding site in an immunoglobulin Fc fragment. Furthermore, methods of preparing fusion proteins by a genetic recombination method using the immunoglobulin Fc region modified as described above are also disclosed in U.S. Pat. Nos. 6,277,375, 6,410,008 and 6,444,792. However, bio-medicines having an immunoglobulin Fc fragment fused thereto are required to overcome cytotoxic problems caused by the effector function inherent of the Fc fragment.